Process for the separation and purification of stereoisomeric hydrophenanthrene carboxylic acids and cyclic homologues thereof



Patented Oct. 14, 1947 PROCESS FOR THE SEPARATION AND PURI- FICATION ()F STEREOISOMERIC HYDRO- PHENANTHRENE CARBOXYLK ACIDS AND; CYCLIC HOMOLOGUESTHEREOF Karl Miescher, Richen, and Jules Beer, Basel. Switzerland, assignors to Ciba Pharmaceutical Products, Inc., Summit. N. J., a corporation of New Jersey No Drawing.

Serial No. 630,117. 1945 According to the present invention stereoisomeric hydrophenanthrene 2 carboxylic acids containing in l-position and, if desired, also in 2-position a hydrocarbon radical. further in 7- position a phenolic hydroxyl or a group convertible thereto, and ring-homologues of such carboxylic acids, are separated and purified by treating Application November 21, 1945,

In Switzerland July 6,

9 Claims. (Cl. 260-520) the acids or their salts with solutions the pH value of which is gradually changed and, if desired, subjecting the acid fractions obtained to further purification and separation treatments.

-' 753 [1933]) and according to applicants investigations has the formula COOH CzHs

The same or similar acids can also be obtained by the action of fused alkali on oestradiol, equilenine, dihydroequilenine and the like (German Patents Nos. 705,852 and 719,572). According to known prescriptions working up is efiected by dissolving the melt in water, saturating the strongly alkaline solution with carbon dioxide for the Purpose of separating the starting material and the like, and precipitating with a mineral acid the portion which has remained in solution.

Whereas a uniform carboxylic acid is thus obtained from oestrone and oestradiol, it has been found that highly impure acid mixtures are formed from still less saturated oestrogenic hormones.

According to the process of German Patent No. 719,572 a carboxylic acid of melting point 120-122 C. is obtained from equiienine having a threshold value of 2.5 y when applied orally to the rat. According to the present process it is advantageous to precipitate the aqueous solution of the melt of equllenine or dihydroequilenine with mineral acid, take up the precipitation in ether and extract the acids formed with sodium carbonate. By adjusting the pH value of the sodium carbonate solution to about 7.5 the inactive by-acids are precipitated and can be separated by filtration. In solution remains a mixture of two stereoisomeric. optically active carboxylic acids. When lowering the pH value to about 5.8 to 6.8, the inactive isomer is precipitated at first which melts in pure state at 2542 56 C. The other highly active isomer is finally obtained by further addition of a mineral acid. In pure state it melts at 161-162 C. The oral threshold value amounts only to 0.05-0.1 y. This acid is therefore at least 25 times as active as that described in German Patent No. 719,572 of melting point -122 C. The latter contains apparently only 4 per cent. of the active portion. The separation of the stereoisomeric carboxylic acids obtained.

for example; according to the processes of patent application Serial No. 542,812, filed June 29,. 1944, and patent application Serial No. 622,092, filed October 12, 1945, can be effected in quite similar manner.

Instead of starting from solutions of the salts of the acid mixtures, the solid acids can also be shaken with aqueous solutions whose pH value is gradually raised, whereupon at first only the strong acids, especially the active acids, enter into solution. In the latter case a pH value of less than 6 is necessary. Incidentally it is remarked that the pH value necessary for the dissolution or filtration is slightly dependent on the applied concentration of the acids.

For adjusting the pH value there can be used. for example, alkaline solutions containing caustic alkali, sodium carbonate or bicarbonate which after saturation with carbon dioxide exhibit a pH value which is, within certain limits, all the higher the stronger the alkali contents are. Buffering can also be brought about by other known methods, for example, by the addition of organic or inorganic acids and/or salts thereof. Quite generally care must be taken that the pH value of the butler solution, depending on its concentration, is lowered by the carboxylic acid which has been added.

The stereoisomeric acids obtained according to the present process are subjected, if desired, to further purification or separation treatments, 1 or example, further reprecipitation, crystallization, conversion into derivatives such as esters or ethers, as well as selective adsorption. These steps. can also be combined.

The following examples illustrate the inven-,

tion:

Example 1 Equilenine is heated to 270-280 C. in 3 portions of 2 grams each time with a mixture of 100 grams of potassium hydroxide and 14 cc. of water. After cooling, the melt is dissolved in water, the

solution isacidified with hydrochloric acid and Carbon dioxide is introduced for several hours. through a solution of this acid mixture in cc. 'of saturated sodium carbonate solution and 300 cc. of water. The precipitate is removed by filtration and the clear filtrate is acidified with mineral acid. The thus precipitated acid mixture is dissolved in 3 cc. of saturated'sodium carbonate solution and 100 cc. of water and the solution formed is again treated with carbon dioxide. A hydroxy-acid of melting point 240-250 C. is precipitated after a short time. For the purpose of purificationit can be converted into a methylether-methylester of melting point 99? C. by treatment with diazomethane andsubsequently with dimethylsulfate in dilute caustic soda solution, which ester, on saponification' and splitting up of the other group, yields the pure. free hydroxy-acid, the iso-bisdehydro-doisynolic acid of melting point 254-256 Cfand having a rotation +33" (in alcohol). It is stereoisomeric with the monocarboxylic acid of melting point 161-162 C. described further below and is inactive in the oestrus test.

The filtrate obtained in the preparation of the iso acid is acidified with mineral acid whereby a colorless powder of melting point 130-140 C. is

precipitated. 0.1-0.2! of which is active in the oestrus t st on rats. For the purpose of further purification also this fraction is converted into the methoxymethylester with diazomethane and dimethylsulfate. This ester is dissolved in petroleum ether. filtered over aluminum oxide and the residue obtained by evaporating the filtrate is recrystallized from methanol. The 1-ethyl-2- carbomethoxy 2 methyl 7 methoxy-l :2:3:4- tetrahydrophenanthrene of melting point 103- 104 C. can be converted practically quantitatively, for example. by heating with 4 grams of pyridine hydrochloride to 160-180 C., into the free 1-ethyl-2-methyl7-hydroxy-l:2:3:4-tetrahydroplienanthrene-Z-carboxylic acid, the normal bisdehydro-doisynolic acid of melting point 161-162 C. and having a ,rotation in alcohol With an oral threshold value of 0.057 on rats lhis compound is the hitherto most active oestroeenic compound in the oestrus test.

Era in p10 2 This acid mixtur is dissolved in 8 cc. of saturated sodium carbonate solution and 200 cc. of water.. lN-tartaric acid solution is added until the pH value amounts to 7.5. The voluminous precipitate thus formed is filtered and the clear filtrate is adjusted to a pH value of between 5.8 and 6.8 with tartaric acid solution. 0.7 to 0.8 gram of crude isobisdehydro-doisynolic acid of melting poi-nt 240-250 C. which can be purified by way of the ether-ester as described in Example 1. By precipitating the filtrate with hydrochloric acidthere is obtained a fraction (0.93 gram) which on the basis of physiological tests has a threshold value of 0.1 and contains 7-8 million international rat units, that is to say nearly the total quantity of active substance contained in the crude product. It can be further purified by crystallization, if desired, after an esterification followed by saponification, whereby the normal bisdehydrodoisynolic acid of melting point 161-162 C. already described in Example 1 is obtained.

Example 3 Dihydroequilenine is heated in 4 portions of 2 grams each time with 100 grams of potassium hydroxide and 14 cc. of water in a nickel crucible for 1 hours to 295-300 C. After cooling, the melt is taken up in water and hydrochloric acid is added to the clear solution. The precipitate is then taken up-in either and the ethereal solution is exhaustively extracted with dilute sodium carbonate solution. After further acidifying the alkaline solution with hydrochloric acid there are obtained 5.4-5.6 grams of a crude dark colored acid mixture.

This mixture is dissolved in 20 cc. of absolute acetone and the solution is added to a hot solution of 5 grams of trisodium phosphate in 200 cc. of Water, which has been adjusted to a pH Value of 8.2-8.4 with a few drops of phosphoric acid. Th whole is cooled and allowed to stand for several hours, the undissolved dark by-acids are separated by filtration, the clear filtrate (pH value of about 7.4) is acidified with mineral acid and 1.9 grams of a crystalline product is thus obtained. It is dissolved in 3 cc. of a saturated sodium carbonate solution and cc. of water and carbon dioxide is introduced thereto for some time. The isobisdehydro-doisynolic acid of melting point 254-256 C. is thus precipitated. The pH value of the solution amounts to about 6.5.

The resulting filtrate is mixed with dilute hydrochloric acid and 0.7 grams of bisdehydrodoisynolic acid is thus obtained, which can be purified oiihand by recrystallization from dilute methyl alcohol.

Example 4 2 grams of dihydroequilenine in a mixture'of -100 grams of potassium hydroxide and 10-15 cc. of water are heated in a nickel crucible for 2 hours to 280 C. After cooling, the reaction mass is taken up in water and the resultant solution acidified and extracted with ether. The acid fractions are extracted from the ethereal solution with dilute sodium carbonate solution and precipitated by means of mineral acid. 1.2-1.4 grams of a crude acid mixture are thus obtaincd.

This mixture is dissolved in dilute caustic soda solution and neutralized with phosphoric acid until an alkaline reaction on phenolphthalein is no longer detected. A phosphate butler soluis precipitated,-

stcreoisomeric bisdehydro-doisynolic acids tion of pH value 5.9 is added until the pH value is lowered to 7.6 whereby 0.8-0.9 gram of an inactive acid fraction is precipitated which is removed by filtration. Practically only the two remain in the filtrate: these can be separated and purified according to the methods described in Examples 2 and 3.

Th inactive acid fractions including. the isobisdehydro-doisynolic acid can also be removed in one single operation by adjusting the alkaline solution of the crude acid mixture immediately to a pH value of 5.8, so that only the normal bisdehydro-doisynolic acid remains dissolved in the filtrate after separating the precipitated fractions.

Instead ofthe hitherto named 7-hydroxy-1z2- dialkylhydrophenanthrene 2 carboxylic acids, also other stereoisorneric hydrophenanthrene-2- carboxylic acids can be obtained in this manner, for example, the 7-hydroxy-1:Z-cyclopentapo- 1:2:3:4 tetrahydrophenanthrene 2 carboxylic acids, the '7-hydroxy-octahydrochrysene-carboxylic acids, further such hydrophenanthrene-2- carboxylic acids which do not contain a hydrocarbon radical in 2-position, for example, the 1- a l k yl 7 hydroxy -1:2z3z4-tetrahydrophenanthrene-2-carboxylic acids, or their ring-homologues.

Example 5 grams of a mixture of synthetic bisdehydrodoisynolic acid (1-ethyl-2-methyl 7 hydroxy- 1:2:3:4 tetrahydrophenanthrene 2 carboxylic acids) of melting point 180-212" 0'. obtained according to patent application Serial No. 542,812, filed June 29, 1944, are dissolved in 17.5 cc. of 2N- caustic soda solution, and the resulting solution which is practically neutral is diluted with a solution of 2-4 grams of sodium carbonate (:1-2 equivalents) in 700 cc. of water. Carbon dioxide is introduced until the solution is saturated and the precipitated inactive racemic iso-bisdehydrodoisynolic acid of melting point 235237 C. is suction-filtered. After acidifying the filtrate the racemic normal bisdehydro-doisynolic acid of melting point 204-205 C. is obtained. 0.1V thereof is active in the oestrus test on rats. The yield is quantitative.

Example 6 2 grams of a mixture of the racemic 1:2-diethyl- 7-hydroxy-1:2:3:4 tetrahydrophenanthrene 2- carboxylic acids (obtained, for example, according to patent application Serial No. 542,812, filed June 29, 1944) are dissolved in 3 cc. of saturated sodium carbonate solution and 30 cc. of water. After saturation with carbon dioxide there is obtained a crystalline precipitation (1 gram) which represents the inactive iso-l 2-diethyl-7-hydroxy- 1:2:3:4 tetrahydrophenanthrene 2 carboxylic acid of melting point 267-269 C. The active stereolsomeric compound of melting point 193-195 C. is obtained in quantitative yield from the filtrate by adding dilute hydrochloric acid.

What we claim is:

1. A process for separating into its constituent isomers a mixture selected from the group consisting of a purified mixture of stereoisomeric hydrophenanthrene-Z-carboxylic acids which contain a hydrocarbon radical in l-position and a phenolic hydroxyl in '7-position, a mixture of the alkali metal salts thereof, and a mixture of ring homologues thereof, which comprises subjecting the said mixture to the action of an excess of 2,429 ,ree 1 slightly acid solvent medium whereby separation into z'wtivo and inactive isomers takes place.

2. A process for separating into its constituent isomers a mixture selected from the group consisting of a purified mixture of stereoisorneric hydrophenanthrcne-2-carboxylic acids which contain a hydrocarbon radical in l-position and a phenolic hydroxyl in 7-position, a mixture of th alkali metal salts thereof, and a mixture of ring homologues thereof, hich comprises subjecting the said mixture to the action of an excess of solvent medium and adjusting the pH of the latter until it is slightly acid by the addition thereto of the corresponding quantity of a solution of complementary pH, whereby separation into active and inactive isomers takes place.

3. The process defined in claim 1 wherein the hydrophenanthrene-Z-carboxylic acids contain a hydrocarbon radical in each of the l-and 2-positions in addition to a phenolic hydroxyl in 7- positiom 4. The process defined in claim 1 wherein the hydrophenanthrene-Z-carboxylic acids are 1:2-dialkyl-7-hydroxyhydrophenanthrene-2- carboxylic acids.

5. A process for separating into its constituent isomers a purified mixture of stereoisomeric 1:2- dialkyl-7-hydroxy-phenanthrene 2 carboxylic acids, which comprises dissolving said mixture in a solvent medium having a pH which is on the alkaline side and successively separating said isomers from the solution by the addition of acidifying reagent until the solution is slightly acid whereupon one isomer separates and, after isolation of the said isomer, adding additional acidifying reagent until the other isomer separates out.

6. A process for separating into its constituents a purified mixture of salts of stereoisomeric 1:2 dialkyl 7 hydroxy-hydrophenanthrene 2 -carboxylic acids which comprises dissolving said mixture in a solvent medium having a' pH which is on the alkaline ide and successively separating said isomers from the solution by the addition of acidifying reagent until the solution is slightly acid whereupon one isomer separates and, after isolation of the said isomer, adding additional acidifying reagent until the other isomer separates out.

'7. A process for separating into its constituent isomers a purified mixture of stereoisomeric 1:2- dialkyl-7-hydroxy-phenanthrene 2 carboxylic acids, which comprises subjecting the said mixture to the action of a solvent of'low initial pH, and adding a pH raising solution until the pH of the mixture is only slightly acid, whereby separation into active and inactive isomers takes place.

8. A process for separating into its constituent isomers a purified mixture of salts of stereoiso meric 1:2-diaikyl-7-hydroxy-phenanthrene 2- carboxylic acids, which comprises subjecting the said mixture to the action of a solvent of low initial pH. and adding a raising solution until the pH of the mixture is only slightly acid.

9. Process for separating the mixture of stereoisomeric l-ethyl-Z-methyl -7- hydroxy 1:213:4- tetrahydrophenanthrene-Z-carboxylic acids obtained by the action of fused alkali on a member selected from the group consisting of equilenine and dihydroequilenine and purif ed from by-acids by adjusting a solution of the mixture of the crude acid fractions in sodium carbonate to a pH value of about 7.5, whereupon precipitation of inactive by-acids occurs, which by-acids are removed by filtration, which comprises lowering the 7 pH value 0! the filtrate to about 5.8 to 6.8, isolatlng the-thus formed 1-ethyl- 2-methyl-7-hydroxy- 1:2 :3:4-tetrahydrophenanthrene 2 carboxyllc acid which melts in pure state at about 254-256 C., acidifying the remaining solution with mineral acid and finally isolating the obtained isomeric l-ethyl 2 methyl -7 hydroxy-1:2:3:4- tetrahydrophenanthrene-2-carboxylic acid which melts in pure state at about 161-162 C.

1 KARL MIESCHER.

JULES HEER.

REFERENCES CITED I The following references are of record in the file of this patent:

Number Number UNITED STATES PATENTS Name Date MacCorquodale et al. Jan. 26, 1937 Foster Jan. 1, 1946 Tomlinson Oct. 31, 1944 Migrdichian Dec. 7, 1943 Walker Nov. 1, 1938 Witzel Jan. 30, 1934 FOREIGN PATENTS Country Date- Great Britain Aug. 8. 1929 

